Adenoviruses
Adenoviruses are nonenveloped icosahedral viruses containing double stranded DNA (dsDNA). Adenoviruses are stable to chemical or physical agents and adverse pH conditions, allowing for prolonged survival. In humans there are over 51 different serotypes. The adenovirus genome is linear, non-segmented dsDNA of approximately 30-38 kilobases in size. The virus is reliant on the host cell for survival and replication. The genome has a terminal 55 kilodalton (kDa) protein associated with each of the 5′ ends of the linear dsDNA. These proteins are used as primers in viral replication and ensure that the ends of the virus linear genome are adequately replicated.
Adenoviral Vectors and Gene Transfer
Adenovirus has become a tool to transfer genes into mammalian cells. Adenoviral vectors have numerous advantages compared to other viral vectors (Roberts, P., et al., Trends in Biotechnol. 1998. 16:35-40): they are rapidly manipulated in vitro, have a moderately high cloning capacity, and can be grown to extremely high titers (Lieber. A., et al., J. Virol. 1999. 73:7835-7841; Hartigan-O'Connor, D., et al., Methods in Enzymol. 2002. 346:224-246). Adenoviruses are currently used in about one fourth of gene therapy clinical trials. Since the early 1990s, the number of publications related to adenoviral vectors has increased exponentially. This number is expected to increase even further, as dozens of new genes are being discovered daily (through the sequencing of genomes), and techniques, such as microarrays are available to screen thousands of genes in a short period of time. Gene transfer techniques, such as adenovirus will be increasingly utilized. Research of adenoviral vectors continues as the requirement for larger cloning capacities increases. However, at least two important disadvantages undermine the use of adenoviral vectors: their construction is a time-consuming process, and adenoviral vectors elicit a strong immune response at high doses in vivo.
Methods for the Construction of Adenoviral Vectors
First-Generation Adenoviral Vectors
First-generation adenoviral vectors are adenoviruses in which an exogenous DNA replaces the E1 region, or in some cases the E3 region of the viral genome.
Several techniques have been developed which facilitate the construction of first-generation adenoviral vectors. They can be classified into three categories.
The first category includes methods based on a homologous recombination between a plasmid carrying the gene of interest, and a viral DNA (Kozarsky, K. and Wilson, J., Curr. Op. Genet. Dev. 1993. 3:499-503; Imler, J., et al., Gene Ther. 1995. 2:263-268). Viral plaques appear on average 10 to 15 days after transfection since this recombination is an inefficient process. Moreover, because the viral DNA is extracted from virions, contamination of the virus preparation is frequent, and time-consuming virus purification by plaque assays must be performed. This entire procedure takes 2 to 3 months.
A second category of methods eliminates these problems by using infectious circular adenoviral DNAs that can replicate in bacteria as plasmids (Bett, A., et al., Proc. Natl. Acad. Sci. USA. 1994. 91:8802-8806). No viral background is obtained since these DNAs are too large to be packaged into viral particles. However, the method still requires a homologous recombination event, which is inefficient, and these circular DNAs are replicated less efficiently than linear viral DNAs. Moreover, these plasmids are unstable in E. coli, and their manipulation is therefore difficult, due to the presence of a 200 base pair long palindrome resulting from the head-to-head joining of both ITRs.
A third category includes methods that reconstitute in a plasmid the entire sequence of the desired recombinant virus (He, T., et al., Proc. Natl. Acad. Sci. USA. 1998. 95:2509-2514; Mizuguchi, H. and Kay, M. Hum. Gene Ther. 1998. 9:2577-2583; Danthinne, X., et al., Gene Ther. 2000. 7(1):80-87). Although their construction has been simplified, these plasmid DNAs are poorly infectious: viral plaques take on average 7 to 10 days to appear, and in some instances require 2 to 3 weeks. Given the fact that the life cycle of a virus is about 24 hours, the generation of virus from plasmid DNA is very slow.
Second-Generation and Other Mutant Adenoviral Vectors
Second-generation adenoviral vectors are first-generation vectors deleted for additional genes involved in viral replication (such as, for example, the E2a, E2b or E4 regions) (Amalfitano, A., J. Virol. 1998. 72:926-933; Gorziglia, M., et al., Virol. 1999. 73:6048-6055). Although no method has been designed specifically for their construction, the same discussion as for the first-generation adenoviral vectors applies: plasmid-based methods are a preferred choice since they eliminate potential viral contaminations, but they suffer from the poor infectivity of the viral DNAs isolated from plasmids.
Plasmid-based approaches are also used to construct other mutant adenoviruses; for example, viruses targeted to specific cell types by substituting heterologous ligands for the fiber knob (Einfeld, D., et al., Proc. Natl. Acad. Sci. USA. 1996. 93:5731-5736). The recovery of such mutant viruses would be difficult, not only because of the low efficiency of recovering virus from bacterial plasmids, but also because the targeted membrane receptor might not be as efficient for virus attachment and internalization as the natural receptor, and altering the fiber may affect virion assembly and stability.
Gutless Adenoviral Vectors
Gutless adenoviral vectors are also referred to as “helper-dependent,” “gutted,” or “high-capacity” adenoviral vectors. They are deleted for the entirety of the viral genome, except for the sequences necessary for replication and packaging. These vectors have two important advantages: first, they can accommodate up to 36 kilobases of exogenous DNA; and second, they are unable to express viral genes and therefore, they should elicit a decreased immune response and a sustained gene expression (Hartigan-O'Connor, D., et al., J. Virol. 1999. 73:7835-7841; Kochanek, S., et al., Proc. Natl. Acad. Sci. USA. 1996. 93:5731-5736; Parks, R., et al., Proc. Natl. Acad. Sci. USA. 1996. 93:13565-13570).
The starting point for the production of a gutless virus is a plasmid DNA that contains the viral ITR's, the packaging signal, and the exogenous DNA. This plasmid generally is linearized and transfected into a cell line with a helper DNA, which provides in trans all the viral products necessary for virus replication. Replication of the helper virus eventually causes lysis of the cells. Unfortunately, the titer of gutless virions is very small compared to the titer of helper virus. Titers of less than 100 particles per milliliter are often obtained on the first passage (Hartigan-O'Connor, D., et al., J. Virol. 1999. 73:7835-7841). To increase the proportion of gutless viruses, the initial lysate must be serially passaged (up to five times), which is very time-consuming. Finally, both gutless and helper viruses must be separated on the basis of their different density on a cesium chloride gradient.
In summary, methods that produce first- and second-generation adenoviral vectors, mutant adenoviruses and gutless viruses, preferentially use bacterial plasmids to generate the recombinant virus. Once transfected into helper cells, these plasmids unfortunately are inefficient in generating the virus; typically, it takes 7 to 10 days and sometimes several weeks to generate viral plaques, or the viral titer is very low (gutless viruses).
Without being limited by theory, the presence of additional nucleotides at the ends of the viral DNA, originating from the restriction site used for linearization of the plasmid, and the absence of the terminal protein, may prevent efficient initiation of DNA replication and may contribute to the low infectivity of these DNAs.
Adenoviral Terminal Protein (TP)
In a virion, the viral DNA is covalently linked to the 55 kDa terminal protein (TP). Genome-linked TP has at least three roles during infection. First, TP determines the sub-nuclear location of viral DNA templates for transcription and replication by binding strongly to the nuclear matrix (Shaack, J., et al., Genes Dev. 1990. 4:1197-1208). Second, TP directly influences DNA replication by altering the structure of linked origin of DNA replication and stabilizing the binding of a pre-terminal protein-viral DNA polymerase complex to its binding site in the viral origin of DNA replication (Pronk, R. and van der Vliet, P. Nucl. Acids Res. 1993. 21:2293-2300). Third, TP may protect the viral DNA against cellular exonucleases (Hay, R., et al., in: Molecular repertoire of adenoviruses II (eds W. Doerfler and P. Bohm), 31-48 (1996)).
The presence of TP at both ends of the viral genome increases its infectivity by two to three orders of magnitude, compared to protease-treated DNA ((van Bergen, B., et al., Nucl. Acids Res. 1983. 11:1975-1988; Pronk, R. and van der Vliet, P. Nucl. Acids Res. 1993. 21:2293-2300; Sharp, P., et al., J. Virology. 1976. 75:442-456; Jones, N. and Shenk, T. Cell. 1978. 13:181-88). Plasmid DNAs are obviously not linked to the adenoviral TP, and in addition, both origins of replication contain a few additional nucleotides originating from the restriction site used to linearize the plasmid. Without being limited by theory, these facts may explain the low infectivity of plasmid DNAs.
Methods for Constructing Adenoviruses using the Terminal Protein
A method designed to construct first-generation adenoviruses with the help of the adenoviral terminal protein, uses a DNA-terminal protein complex purified from virions (Miyake, S., et al., Proc. Natl. Acad. Sci. USA. 1996. 93:1320-1324). This viral DNA is digested extensively using a specific restriction enzyme, and transfected with a plasmid DNA containing the gene of interest into helper cells. This technique allows recovery of hundreds of plaques. However, it still requires the very inefficient homologous recombination event in helper cells and 30% of the plaques are negative. The very time-consuming process of screening for the recombinant virus is therefore still required.
In another approach, a stable cell line was generated that expresses both the adenoviral DNA polymerase and the pre-terminal protein (Hartigan-O'Connor, D., et al., J. Virol. 1999. 73:7835-7841). This cell line was shown to increase the efficiency of generation of recombinant viruses from plasmids. Unfortunately, because it is derived from the “293” cell line, it cannot prevent the formation of replication-competent adenoviruses (RCA) from first-generation adenoviral vectors. Therefore, time-consuming virus purification and tests for the presence of RCAs must be performed if the virus is used in clinical trials. Moreover, cells lines expressing the terminal protein are difficult to establish and to maintain because of the toxicity of the terminal protein. This complicates the construction of helper cell lines that express additional heterologous genes, coding for instance for a membrane receptor to which a recombinant virus is targeted.
In another approach, gutted Ad vectors from plasmid-derived substrates or from synthetic TP-linked substrates made in vitro have been attempted (Hartigan-O'Connor, D., et al., Hum. Gene Ther. 2002. 13:519-531). Efficient rescue required cotransfection of gutted and helper genomes with identical origins of replication. Cotransfection of plasmid-derived substrates was 30 times more efficient than transfection/infection. Linkage of gutted vector genomes to TP and expression of Cre recombinase further increased rescue efficiency.
Gutless adenovirus vectors constitute a promising tool for gene transfer because of their unique transgene capacity (up to 36 kb), prolonged persistence and their reduced cytotoxicity and immunogenicity compared to first-generation vectors. One of the major hurdles in gutless adenovirus vectors production is the difficulty in large-scale production; a difficulty that has contributed to the lack of successful clinical trials.
Current methods for generating gutless adenovirus vectors start with the rescue of the gutless virus by transfecting plasmid DNA into helper cells. However, plasmid DNA is poorly infectious, and the initial titers obtained upon transfection are generally too low for an efficient amplification of the gutless virus. The process necessitates a series of time-consuming and labor-intense virus passages, with the consequence that the gutless virus preparations are often contaminated with products of recombination. It becomes clear that the lower the number of gutless viral particles produced in rescue, the higher the number of passages required before purification. Therefore a technique which can enhance the rescue of virus from plasmid DNA is needed.
The present disclosure addresses this problem as it provides a method for binding adenovirus terminal protein to linear DNA.
To improve the critical step of gutless virus rescue, the present invention has utilized the adenovirus terminal protein. In the virion, the linear genomic DNA is linked at each end to a 55 kDa terminal protein (TP). To increase the infectivity of plasmid DNAs, a method has been disclosed to link the TP to plasmid DNA ends. Because the TP is bound to DNA in a very specific way, which would be very difficult to achieve in routine, the TP is purified from virions as a complex with a short stretch of DNA (the inverted terminal repeat—ITR), that is linked to the ends of linearized plasmid DNA by a DNA ligation reaction. This methodology is referred to hereafter as the “TP-ITR” method.
The use of the TP to generate gutless adenovirus vectors from plasmid DNA translates both in a shorter virus recovery time and in higher virus yields when compared to methods that use plasmid DNA devoid of TP. By considerably improving the first rescue step in the construction of gutless vectors, the TP-ITR method decreases the number of passages that are required to obtain high-titer virus preparations. Thus the method speeds up the process, and also leads to a superior product by decreasing the possibility of gutless vector recombination.
There are at least four advantages of using the TP-ITR method for producing gutless adenovirus. First, the binding of the terminal protein (TP) to the linearized plasmid DNA used to generate the gutless virus is consistently reproducible and efficient. The TP is indeed provided as a covalent complex with the inverted terminal repeat (ITR), which is linked to the gutless genome by a DNA ligation. Because both DNAs have complementary sticky ends which are not symmetrical, the formation of TP-ITR dimers or the recircularization of the gutless genome is prohibited, and ligation of the TP-ITR to the gutless genome can be completed at almost 100% efficiency in just one hour, using a T4 DNA ligase.
A second advantage is that very high titers of gutless virus can be obtained upon transfecting the gutless plasmid and helper plasmid DNAs into helper cells (passage “0”). Utilizing 2 μg of gutless plasmid can generate up to 1.5×108 gutless virus particles, that is at least 2800-fold greater than methods that do not incorporate the TP-ITR.
A third advantage is that the virus suspension obtained from passage “0” is virtually free of helper virus. Indeed the genome of the helper virus used in the transfection step can be deleted from the packaging signal, and as a result can share no homology with the genome of the gutless virus (except for the ITR), and would not generate replication-competent adenovirus (RCA) by homologous recombination.
Finally the TP-ITR complex is purified from the helper virus itself, which is used downstream to amplify the virus (passages 1, 2, . . . ). This drops any concern about potential contamination of the TP-ITR preparations with the source virus, since this latter is added anyway to the gutless virus extract later during the amplification.